1. Field of the Invention
The present invention relates to the use of materials for immobilizing oligonucleotides and other biomolecules for the construction of genosensors and other array-based systems. The use of the materials according to the present invention allows for the attachment of previously synthesized oligonucleotides and other biomolecules at high ligand densities with low background fluorescence and non-specific adsorption.
2. Description of the Prior Art
Nucleic acid hybridization assays have been used extensively in molecular biology to establish the sequence similarity of populations of nucleic acids. Hybridization is simply the annealing or pairing of single stranded nucleic acid molecules (DNA or RNA) to form double strands. The most common modern technique employing hybridization is the Southern blot hybridization, invented by Dr. E. Southern (1) in which a set of unknown target DNA molecules is immobilized on a membrane and a labeled probe DNA molecule in solution is used to bathe the membrane under conditions where complementary molecules will anneal. In an analogous technique called Northern blot hybridization (2,3). RNA molecules immobilized on membranes are the targets. The labeled probe DNA used in the liquid phase can be as short as 10-20 nucleotides: probes are usually labeled with radioisotopes, although other reporter groups (e.g., fluorescein, biotin, etc.) can be used. Reverse blot hybridization employs the opposite approach. Instead of immobilizing unknown DNAs, a set of well defined DNA probes are immobilized on a solid surface, and unknown labeled "target" DNA is present in the liquid phase. Therefore, in reverse hybridizations a large number of probes can be used with single target molecules. By decoding the hybridization pattern of the unknown DNA to positions of known sequence on the solid phase array, sequence information from several positions of the unknown target DNA can be obtained.
The first interest in reverse hybridization was spurred by the idea that oligonucleotide probes could be used to sequence unknown DNA samples by observing the pattern of positive hybridization to all possible oligonucleotide sequences of a fixed length (refs. 4-8). The speed of the assay and its potential for automation compared with existing sequencing techniques has generated much excitement in the genome sequencing community. However, the ability to assay shorter known DNA segments for the sequence alterations by reverse hybridization appears to be a more practical application at present.
There are two fundamental ways of generating oligonucleotide arrays: the oligonucleotides may be synthesized on the solid phase in their respective positions, or they may be synthesized apart from the surface of the array matrix and attached later. The former method has been successfully achieved in several different ways. The first reverse hybridization arrays to be synthesized in situ were made by Dr. Southern, using glass modified with an aliphatic poly(ether) linker as a solid support (9).
Alternatively, unique sets of photosensitive protecting groups have also been used during DNA synthesis to selectively build reverse hybridization arrays. In this photolithographic technique, light is used to unmask specific hydroxyl groups within the growing array to sequentially add the desired bases (10).
The immobilization of previously synthesized oligonucleotides has been approached in different ways. In general, the attachment of standard oligonucleotides to unmodified glass or plastic surfaces is inefficient. For this reason, most investigators trying to immobilize oligonucleotides modify them with molecules that promote adsorption or enable covalent attachment to the support. Oligonucleotides modified with bovine serum albumin adsorb passively to microliter plates designed to bind protein molecules (11). Biotinylated oligonucleotides bind tightly to plates or beads that are coated with avidin or streptavidin. Oligonucleotides with polythymidylate tails have been photochemically crosslinked to nylon (12). More recently, oligonucleotides with terminal amino (13,14) or methyluridine (15) groups have been covalently crosslinked to compatible reactive groups on multi-well plate surfaces.
Both approaches to array construction have different advantages. Synthesis in situ does not involve handling thousands or tens of thousands of independent oligonucleotides, each of which must be produced on a scale that far exceeds that which is required for the array. In contrast, the ability to freely arrange the members of an array after oligonucleotide synthesis is only possible with pre-synthesized oligonucleotides. Matson et al., has recently published two papers on the development of surface chemistry for reverse hybridization arrays and on the use of arrays to detect DNA sequence repeats in a variety of target DNA's. In the first paper (16), the authors examine the feasibility of using polypropylene as a support for the construction of reverse hybridization arrays. In addition to being easy to handle, a good support must possess several important properties; it must be stable under the conditions of harsh organic synthesis encountered during DNA synthesis, while having low non-specific binding of DNA during aqueous hybridization. It is also advantageous for the support to have low fluorescent background for sensitive non-radioactive detection. Native polypropylene possesses all of these qualities, but DNA synthesis cannot be done on the unmodified polymer. Matson et al. used radio-frequency plasma discharge in the presence of ammonia gas to aminate the polypropylene surface. The amino group offers a sufficiently active nucleophile to promote nucleic acid base coupling to the support using the conventional CED-phosphoramidite chemistry employed to synthesize oligonucleotides on modern DNA synthesizers including the Beckman Oligo 1000. Oligonucleotides synthesized on aminated polypropylene when released from the support were found to be of excellent quality, a prerequisite for use in further hybridization experiments.
While most hybridization assays rely on detecting signals from DNA molecules while they are still bound to their targets, the desorption (dissociation of "melting") profile may also be used to differentiate complementary and mismatched hybridizations. Matson et al. devised an elution system using a modified flow-cell and radioisotope detector with a Beckman Model 126 HPLC gradient system. Small pieces of hybridized polypropylene membrane or threads on which a single oligonucleotide probe sequence was synthesized were placed in the flow cell and the previously annealed target DNA was slowly eluted using a decreasing linear salt gradient. Using test oligonucleotide targets, the dissociation of a short target from the complementary oligonucleotide covalently bound to the support was slightly faster for a target with a single nucleotide mismatch and much faster for a target containing a two nucleotide detection. More importantly, a target derived from a cystic fibrosis patient was shown to have a dramatically different elution profile than a target from a normal individual. In addition to obtaining information on the dissociation process, the dynamic hybridization analysis system is also a way that designers of reverse hybridization arrays can optimize the buffer conditions to obtain binding of perfectly matched nucleic acid sequences.
The second paper, done in collaboration with Drs. Manfred Wehnert and Thomas Caskey from Baylor College of Medicine (17), uses reverse hybridization arrays to identify the presence of dinucleotide and trinucleotide repeats in PCR amplified DNA samples. These repeats, along with tetranucleotide repeats, comprise the class of DNA sequences called short tandem repeats or STR's which are dispersed throughout the human genome. STR's are generally found in regions of the genome that do not code for proteins and they often vary in copy number and therefore overall length between individuals. These variations in size (STR polymorphisms) usually do not cause any harmful effects. By comparing the size of repeats at specific locations in divergent individuals, the polymorphisms can be used to track the inheritance of specific gene segments and in this way they can be used just like restriction fragment length polymorphisms (18) to predict disease status to map unknown genes and to identify individuals.
However, in a few important chromosomal locations where the repeats have been found within genes, an increase in repeat size has been shown to cause disease. Huntington's Disease (19) and the fragile X mental retardation syndrome (20-22) are two important examples. Thus, it is useful to identify molecular clones of genomic DNA that carry these repeated sequences. Wehnert et al. demonstrates that using both DNA sequences known to contain specific dinucleotide and trinucleotide repeats, and a cosmid alone that contains unknown repeats as targets, one can obtain hybridization signals that reflect the presence of these repeats.
As mentioned above, there are many supports that will bind nucleic acids. However, few of these are suitable for the attachment of small oligonucleotides (10mer to 20mer) at sufficient ligand density to be useful for hybridization and detection of complementary target nucleic acids. Moreover, commercially available DNA binding membranes currently available suffer from high background fluorescence and non-specific adsorption. A need therefore still exists for support materials which bind small oligonucleotides which have low intrinsic background fluorescence and low non-specific adsorption properties.